Phosphorylation of Streptococcus salivarius lactose permease (LacS) by HPr(His ~ P) and HPr(Ser-P)(His ~ P) and effects on growth

The oral bacterium Streptococcus salivarius takes up lactose via a transporter called LacS that shares 95% identity with the LacS from Streptococcus thermophilus, a phylogenetically closely related organism. S. thermophilus releases galactose into the medium during growth on lactose. Expulsion of galactose is mediated via LacS and stimulated by phosphorylation of the transporter by HPr(His approximately P), a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). Unlike S. thermophilus, S. salivarius grew on lactose without expelling galactose and took up galactose and lactose concomitantly when it is grown in a medium containing both sugars. Analysis of the C-terminal end of S. salivarius LacS revealed a IIA-like domain (IIA(LacS)) almost identical to the IIA domain of S. thermophilus LacS. Experiments performed with purified proteins showed that S. salivarius IIA(LacS) was reversibly phosphorylated on a histidine residue at position 552 not only by HPr(His approximately P) but also by HPr(Ser-P)(His approximately P), a doubly phosphorylated form of HPr present in large amounts in rapidly growing S. salivarius cells. Two other major S. salivarius PTS proteins, IIAB(L)(Man) and IIAB(H)(Man), were unable to phosphorylate IIA(LacS). The effect of LacS phosphorylation on growth was studied with strain G71, an S. salivarius enzyme I-negative mutant that cannot synthesize HPr(His approximately P) or HPr(Ser-P)(His approximately P). These results indicated that (i) the wild-type and mutant strains had identical generation times on lactose, (ii) neither strain expelled galactose during growth on lactose, (iii) both strains metabolized lactose and galactose concomitantly when grown in a medium containing both sugars, and (iv) the growth of the mutant was slightly reduced on galactose.     

HPr(His approximately P)-mediated phosphorylation differently affects counterflow and proton motive force-driven uptake via the lactose transport protein of Streptococcus thermophilus

The lactose transport protein (LacS) of Streptococcus thermophilus has a C-terminal hydrophilic domain that is homologous to IIA protein and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The IIA domain of LacS is phosphorylated on His-552 by the general energy coupling proteins of the PTS, which are Enzyme I and HPr. To study the effect of phosphorylation on transport, the LacS protein was purified and incorporated into liposomes with the IIA domain facing outwards. This allowed the phosphorylation of the membrane-reconstituted protein by purified HPr(His approximately P) of S. thermophilus. Phosphorylation of LacS increased the V(max) of counterflow transport, whereas the V(max) of the proton motive force (delta p)-driven lactose uptake was not affected. In line with a range of kinetic studies, we propose that phosphorylation affects the rate constants for the reorientation of the ternary complex (LacS with bound lactose plus proton), which is rate-determining for counterflow but not for delta p-driven transport.     

The HPr(Ser) Kinase of Streptococcus salivarius: Purification, Properties, and Cloning of the hprK Gene

In gram-positive bacteria, HPr, a protein of the phosphoenolpyruvate:sugar phosphotransferase system, is phosphorylated on a serine residue at position 46 by an ATP-dependent protein kinase. The HPr(Ser) kinase of Streptococcus salivarius ATCC 25975 was purified, and the encoding gene (hprK) was cloned by using a nucleotide probe designed from the N-terminal amino acid sequence. The predicted amino acid sequence of the S. salivarius enzyme showed 45% identity with the Bacillus subtilis enzyme, the conserved residues being located mainly in the C-terminal half of the protein. The predicted hprK gene product has a molecular mass of 34,440 Da and a pI of 5.6. These values agree well with those found experimentally by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, molecular sieve chromatography in the presence of guanidine hydrochloride, and chromatofocusing using the purified protein. The native protein migrates on a Superdex 200 HR column as a 330,000-Da protein, suggesting that the HPr(Ser) kinase is a decamer. The enzyme requires Mg²⁺ for activity and functions optimally at pH 7.5. Unlike the enzyme from other gram-positive bacteria, the HPr(Ser) kinase from S. salivarius is not stimulated by FDP or other glycolytic intermediates. The enzyme is inhibited by inorganic phosphate, and its K_ms for HPr and ATP are 31 μM and 1 mM, respectively.     

The constant gene orf14.9, which belongs to the variable eps (exopolysaccharide) cluster, is involved in the cell growth of Streptococcus thermophilus

In Streptococcus thermophilus, the eps clusters involved in exopolysaccharide (EPS) biosynthesis are very polymorphic, nevertheless they all contain a highly conserved sequence corresponding to that of orf14.9. This open reading frame (ORF) is transcribed in a reverse direction with respect to eps genes. Amino acid sequence analysis showed a possible transmembrane location of the putative Orf14.9 protein but did not permit a proposed function. Insertional mutants of orf14.9 were obtained in strains NST2280 and A054 of S. thermophilus. EPS yields of these mutants are similar to those of their respective wild strains, suggesting that orf14.9 does not modify the quantity of produced EPS. Growth parameter determination for wild strains and their respective mutants showed that orf14.9 is involved in the cell growth of S. thermophilus.     

Effect of chloramphenicol on the growth and viability of exponentially growing mouse leukemic cells (L5178Y)

Two effects of chloramphenicol on mouse leukemic cells (L5178Y) are described. The drug induces a prolongation of the cell cycle (reversible effect). The degree of prolongation is directly proportional to the concentration of the drug. The effect is observed only in the presence of chloramphenicol and cells return to normal cell-growth kinetics when the drug is removed from the culture medium. Chloramphenicol also kills a portion of the cell population immediately (irreversible effect). Data are presented which suggest that chloramphenicol is toxic to cells in the G2 phase of the cell cycle.     

Synthesis of peptidoglycan in the form of soluble glycan chains by growing protoplasts (autoplasts) of Streptococcus faecalis.

Protoplasts (autoplasts) of Streptococcus faecalis were produced by the action of native autolytic N-acetylmuramidase in the absence of added peptidoglycan hydrolases and were grown in osmotically stabilized medium containing L-[3H]lysine and D-[14C]alanine. To reduce the level of muralytic hydrolysis of glycan chains during growth, heat-inactivated cell walls were added to the medium to bind autolytic enzyme, and tetracycline (1 mug/ml) was added to inhibit further enzyme synthesis. Under these conditions, protoplasts synthesized newly labeled peptidoglycan in the form of soluble, infrequently peptide cross-linked glycan chains which were released into the supernatant medium. These relatively large glycan chains were not transferred to exogenously added cell walls.     

The ATP-binding cassette transporter ABCG2 (BCRP), a marker for side population stem cells, is expressed in human heart.

Efforts to improve severely impaired myocardial function include transplantation of autologous hematopoietic side population (SP) stem cells. The transmembrane ABC-type (ATP binding cassette) half-transporter ABCG2 (BCRP) serves as a marker protein for SP cell selection. We have recently shown that other ABC transport proteins such as ABCB1 and ABCC5 are differentially expressed in normal and diseased human heart. Here we investigated localization and individual ABCG2 expression in 15 ventricular (including 10 cardiomyopathic) and 51 auricular heart tissue samples using immunohistochemistry, confocal laser scanning fluorescence microscopy, and real-time RT-PCR. Individual genotypes were assigned using PCR-restriction fragment length polymorphism (RFLP) analysis and subsequently correlated to ABCG2 mRNA levels. ABCG2 was localized in endothelial cells of capillaries and arterioles of all samples. Ventricular samples from cardiomyopathic hearts exhibited significantly increased levels of ABCG2 mRNA (ABCG2/18S rRNA: 1.08 +/- 0.30 x 10(-7); p=0.028 (dilative cardiomyopathy) and 1.16 +/- 0.46 x 10(-7); p=0.009 (ischemic cardiomyopathy) compared with 0.44 +/- 0.26 x 10(-7) in nonfailing hearts). The individual haplotypes were not associated with altered mRNA expression. ABCG2 is variably expressed in endothelial cells of human heart, where it may function as a protective barrier against cardiotoxic drugs such as anthracyclines or mitoxantrone. ABCG2 expression is induced in dilative and ischemic cardiomyopathies.     

Molecular structure,solution chemistry and biological properties of the novel [ImH][trans-IrCl(4)(Im)(DMSO)], (I) and of the orange form of [(DMSO)(2)H][trans-IrCl(4)(DMSO)(2)], (II), complexes

The new iridium(III) complex, imidazolium[trans(DMSO,imidazole)tetrachloroiridate(III)], (I) (DMSO=dimethyl sulfoxide), and the orange form of [(DMSO)(2)H][trans(DMSO)(2)tetrachloroiridate(III)], (II) have been prepared and characterized, both in the solid state and in solution, by X-ray diffraction and by various physicochemical techniques. Single crystal X-ray diffraction studies point out that complex (II) is isomorphous to the ruthenium(III) analogue, [(DMSO)(2)H][trans-RuCl(4)(DMSO)(2)], (III). Crystallographic data are the following: a=16.028(2) A, b=24.699(3) A, c=8.262(1) A, in space group Pbca (Z=8) for (imidazolium)[trans(DMSO,imidazole)tetrachloroiridate(III)], (I); and a=9.189(2) A, b=16.511(4) A, c=14.028(3) A, beta=100.82(2) degrees in space group P2/n (Z=4) for [(DMSO)(2)H][trans(DMSO)(2)tetrachloroiridate(III)], (II). Visible absorption spectra show that both complexes are stable for several days, at pH 7.4, at room temperature. No significant chloride hydrolysis is observed, even at high temperature (70 degrees C), over 24 h. The extreme stability of these iridium(III) complexes within a physiological buffer was further assessed by (1)H NMR; in addition, cyclic voltammetry measurements evidenced a high stability of the oxidation state +3. Preliminary biological studies show that both complexes do not bind appreciably bovine serum albumin nor inhibit significantly the proliferation of representative human tumor cell lines, suggesting that hydrolysis of coordinated chlorides is a crucial feature for the biological properties and the antitumor activity of the parent ruthenium(III) complexes.     

The (Na,K)-ATPase of Friend erythroleukemia cells is phosphorylated near the ATP hydrolysis by an endogenous membrane-bound kinase

Friend murine erythroleukemia cells (MEL cells) contain a cAMP-independent protein kinase which phosphorylates the 100,000-Da catalytic subunit of the (Na,K)-ATPase both in living cells and in the purified plasma membrane (Yeh, L.-A., Ling, L., English, L., and Cantley, L. (1983) J. Biol. Chem. 258, 6567-6574). We have taken advantage of the selective phosphorylation of the 100,000-Da subunit in purified plasma membranes and the similarity between the proteolysis patterns of the MEL cell and dog kidney (Na,K)-ATPase to map the site of kinase phosphorylation on the MEL cell enzyme. The chymotryptic and tryptic cleavage sites of the dog kidney (Na,K)-ATPase have previously been located (Castro, J., and Farley, R. A. (1979) J. Biol. Chem. 254, 2221-2228). The 100,000-Da catalytic subunits of the dog kidney and MEL cell enzymes were specifically labeled at the active site aspartate residue by incubation with (32P)orthophosphate in the presence of Mg2+ and ouabain. Digestion of these two enzymes with chymotrypsin or trypsin revealed similar active site aspartate containing proteolytic fragments indicating a similar structure for the two enzymes. Chymotryptic digestions of MEL cell (Na,K)-ATPase labeled in vitro with [gamma-32P]ATP localize the region of kinase phosphorylation to within a 35,000-Da peptide derived from the middle of the 100,000-Da subunit. Tryptic digestion of the MEL cell plasma membranes degraded the 100,000-Da subunit to an NH2-terminal 43,000-Da peptide which contained the active site aspartate but which did not contain the kinase-labeled region. These results further locate the region of kinase phosphorylation to the COOH-terminal half of the 35,000-Da chymotryptic peptide. This location places the site of phosphorylation between the active site aspartate residue which accepts the phosphate of ATP during turnover and an ATP-binding site which has previously been located by labeling with fluorescein 5'-isothiocyanate (Carilli, C. T., Farley, R. A., Perlman, D. M., and Cantley, L. C. (1982) J. Biol. Chem. 257, 5601-5606). Phosphorylation of the (Na,K)-ATPase in this region may serve to regulate the activity of this enzyme.     

Expression of fra(X)(q28) is Suppressed in man-mouse hybrid cells

Summary The fate of fra(X) was followed after fusion of cells donated by a male fra(X) carrier with mouse A9 cells. Suppression of the fragile site was found in the hybrids as well as in human cells co-cultivated with mouse cells.     

Expression of the activity of cystine/glutamate exchange transporter,system x(c)(-), by xCT and rBAT

The expression of the activity of cystine/glutamate exchange transporter, designated system x(c)(-), requires two components, xCT and 4F2 heavy chain (4F2hc) in Xenopus oocytes. rBAT (related to b(0,+) amino acid transporter) has a significant homology to 4F2hc and is known to be located in the apical membrane of epithelial cells. To determine whether xCT can associate with rBAT and express the activity of system x(c)(-), xCT, and rBAT were co-expressed in Xenopus oocytes and in mammalian cultured cells. In the oocytes injected with rBAT cRNA alone, the activities of cystine and arginine transport were induced, indicating that the system b(0,+)-like transporter was expressed by associating the exogenous rBAT with an endogenous b(0,+)AT-like factor as reported previously. In the oocytes injected with xCT and rBAT cRNAs, the activity of cystine transport was further induced. This induced activity of cystine transport was partially inhibited by glutamate or arginine and completely inhibited by adding both amino acids. In these oocytes, the activity of glutamate transport was also induced and it was strongly inhibited by cystine. In NIH3T3 cells transfected with xCT cDNA alone, the activity of cystine transport was significantly increased, and in the cells transfected with both xCT and rBAT cDNAs, the activity of cystine transport was further enhanced. The enhanced activity was Na(+)-independent and was inhibited by glutamate and homocysteate. These results indicate that rBAT can replace 4F2hc in the expression of the activity of system x(c)(-) and suggest that system x(c)(-) activity could be expressed in the apical membrane of epithelial cells.     

Reduced apo-fumarate nitrate reductase regulator (apoFNR) as the major form of FNR in aerobically growing Escherichia coli

Under anoxic conditions, the Escherichia coli oxygen sensor FNR (fumarate nitrate reductase regulator) is in the active state and contains a [4Fe-4S] cluster. Oxygen converts [4Fe-4S]FNR to inactive [2Fe-2S]FNR. After prolonged exposure to air in vitro, apoFNR lacking a Fe-S cluster is formed. ApoFNR can be differentiated from Fe-S-containing forms by the accessibility of the five Cys thiol residues, four of which serve as ligands for the Fe-S cluster. The presence of apoFNR in aerobically and anaerobically grown E. coli was analyzed in situ using thiol reagents. In anaerobically and aerobically grown cells, the membrane-permeable monobromobimane labeled one to two and four Cys residues, respectively; the same labeling pattern was found with impermeable thiol reagents after cell permeabilization. Alkylation of FNR in aerobic bacteria and counting the labeled residues by mass spectrometry showed a form of FNR with five accessible Cys residues, corresponding to apoFNR with all Cys residues in the thiol state. Therefore, aerobically growing cells contain apoFNR, whereas a significant amount of Fe-S-containing FNR was not detected under these conditions. Exposure of anaerobic bacteria to oxygen caused conversion of Fe-S-containing FNR to apoFNR within 6 min. ApoFNR from aerobic bacteria contained no disulfide, in contrast to apoFNR formed in vitro by air inactivation, and all Cys residues were in the thiol form.     

Identification of lactotropes (PRL cells), somatotropes (GH cells), corticotropes (ACTH cells) and thyrotropes (TSH cells) in the pituitary gland of the musk shrew, Suncus murinus L. (Insectivora), by immunohistochemistry

By means of immunohistochemistry, lactotropes, somatotropes, corticotropes and thyrotropes in the pituitary of the male musk shrew, Suncus murinus L., were identified at the electron-microscopic level. Lactotropes were classified into three types: type I containing large (200-450 nm in diameter) round secretory granules, type II with medium-sized (150-250 nm in diameter) ones and type III with small (50-150 nm in diameter) ones. Somatotropes were also classified into type I somatotropes that contain large (450 nm in diameter) spherical secretory granules and type II somatotropes containing comparatively small (300 nm in diameter) round granules. Both type I and II somatotropes were small and sometimes contained rod-shaped granules. Corticotropes were round or oval cells with round secretory granules in various densities and sizes (150-500 nm in diameter) scattered all over the cytoplasm. Thyrotropes were angular or polyhedral cells containing electron-transparent round secretory granules (200-300 nm in diameter) and large irregularly shaped granules with a maximum diameter of about 1,500 nm. Each type of the cells may be distinguished by its respective ultrastructural characteristics alone without the aid of immunohistochemistry.     

The inhibitory spectrum of thermophilin 9 from Streptococcus thermophilus LMD-9 depends on the production of multiple peptides and the activity of BlpG(St), a thiol-disulfide oxidase

The blp(St) cluster of Streptococcus thermophilus LMD-9 was recently shown to contain all the genetic information required for the production of bacteriocins active against other S. thermophilus strains. In this study, we further investigated the antimicrobial activity of S. thermophilus LMD-9 by testing the susceptibility of 31 bacterial species (87 strains). We showed that LMD-9 displays an inhibitory spectrum targeted toward related gram-positive bacteria, including pathogens such as Listeria monocytogenes. Using deletion mutants, we investigated the contribution of the three putative bacteriocin-encoding operons blpD(St)-orf2, blpU(St)-orf3, and blpE(St)-blpF(St) (bac(St) operons) and of the blpG(St) gene, which encodes a putative modification protein, to the inhibitory spectrum and immunity of strain LMD-9. Our results present evidence that the blp(St) locus encodes a multipeptide bacteriocin system called thermophilin 9. Among the four class II bacteriocin-like peptides encoded within the bac(St) operons, BlpD(St) alone was sufficient to inhibit the growth of most thermophilin 9-sensitive species. The blpD(St) gene forms an operon with its associated immunity gene(s), and this functional bacteriocin/immunity module could easily be transferred to Lactococcus lactis. The remaining three Bac(St) peptides, BlpU(St), BlpE(St), and BlpF(St), confer poor antimicrobial activity but act as enhancers of the antagonistic activity of thermophilin 9 by an unknown mechanism. The blpG(St) gene was also shown to be specifically required for the antilisteria activity of thermophilin 9, since its deletion abolished the sensitivities of most Listeria species. By complementation of the motility deficiency of Escherichia coli dsbA, we showed that blpG(St) encodes a functional thiol-disulfide oxidase, suggesting an important role for disulfide bridges within thermophilin 9.     

Isaria macroscyticola,a rare entomopathogenic species on Cydnidae (Hemiptera), is a synnematous form and synonym of Purpureocillium lilacinum (Ophiocordycipitaceae)

Isaria macroscyticola is a hypocrealean pathogen of Cydnidae first described by Dr. Yosio Kobayasi in 1941. Since then, records of this species have been limited, and therefore no cultures have been established and its phylogenetic position is unknown. Recently, synnemata on Macroscytus japonensis were collected from Kanagawa Prefecture, Japan. These were identified as I. macroscyticola based on the morphologies of the synnemata, conidiophores, and conidia. Since cultures established by conidial isolations exhibited pale purplish colonies and Acremonium-like synanamorph, this species was suggested to belong to Purpureocillium (Ophiocordycipitaceae) rather than Isaria (Cordycipitaceae). The sequence similarities of the internal transcribed spacer region and 28S of nuclear ribosomal DNA and the elongation factor 1-alpha gene sequences of I. macroscyticola and P. lilacinum were 99–100%. Furthermore, I. macroscyticola was phylogenetically nested within the P. lilacinum clade. Accordingly, we conclude that I. macroscyticola is a synnematous form and synonym of P. lilacinum.     

Synthesis and characterization of the biological activity of the cisplatin analogs, cis-PtCl(2)(dexrazoxane) and cis-PtCl(2)(levrazoxane), of the topoisomerase II inhibitors dexrazoxane (ICRF-187) and levrazoxane (ICRF-186)

The individual stereoisomers cis-PtCl(2)(dexrazoxane) and cis-PtCl(2)(levrazoxane) were synthesized and their structures were determined by X-ray crystallography. Dexrazoxane and levrazoxane inhibit cell growth because they are strong catalytic inhibitors of DNA topoisomerase II, whereas cisplatin acts through the formation of DNA cross-links. It was hypothesized that platinum(II) complexes of dexrazoxane and levrazoxane would retain both activities and yield drugs with a dual mode of action. Both cis-PtCl(2)(dexrazoxane) and cis-PtCl(2)(levrazoxane) inhibited Chinese hamster ovary cell growth, but more weakly than dexrazoxane and levrazoxane did. Based on their weak topoisomerase II inhibitory activity, it was concluded that these compounds did not inhibit cell growth by targeting topoisomerase II. A comparison of the conformation of cis-PtCl(2)(dexrazoxane) to that of dexrazoxane bound to the dimer interface of topoisomerase II showed that the highly constrained cis-PtCl(2)(dexrazoxane) was in a highly unfavorable conformation for binding. Neither of the platinum complexes were able to cross-link DNA. Thus the cell growth inhibitory activity of these complexes was also not likely due to any cisplatin-type cross-linking activity.     

Gastrin releasing peptide (GRP) is present in a GRP(1-27) form in anterior pituitary cells of the guinea pig

Immunohistochemical and chromatographic studies were performed on the guinea pig anterior pituitary gland with an antiserum recognizing an epitope within the gastrin releasing peptide (GRP) carboxyterminal amino acid sequence Val-Gly-His-Leu-Met-NH2. Within the anterior pituitary gland GRP-like immunoreactive cells were identified. The GRP-like immunoreactive cells were distributed heterogenously in the gland, predominantly located in ventral aspects of the anterior pituitary. Intracellularly, the immunoreactivity elements were identified as granula-like structures in the cytoplasma. To further characterize the peptide displaying GRP-like immunoreactivity within the pituitary cells, the GRP-like substances were analyzed by radioimmunoassay and gel filtration chromatography. Using this analytical approach it was determined that the guinea pig pituitary extract contained a peptide with characteristics similar to that of authentic porcine GRP(1-27). Only trace amounts of smaller C-terminal fragments were identified. These results indicate, in contrast to findings in other tissues, the GRP(1-27) is not further degraded into smaller peptide fragments.     

The effect of 24R,25-(OH)(2)D(3) on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1alpha,25-(OH)(2)D(3) is mediated by phospholipase C

1alpha,25-(OH)(2)D(3) regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)(2)D(3) regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24R,25-(OH)(2)D(3) and determined the role of phospholipases D, C, and A(2) as well as G-proteins in mediating the effects of vitamin D(3) metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)(2)D(3). Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)(2)D(3). This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)(2)D(3) on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1alpha,25-(OH)(2)D(3)-dependent PKC. Inhibition of PI-PLC blocked the 1alpha,25-(OH)(2)D(3)-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA(2) with melittin inhibited basal and 24R,25-(OH)(2)D(3)-stimulated PKC in RC cells and stimulated basal and 1alpha,25-(OH)(2)D(3)-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)(2)D(3) on PKC, whereas GDPbetaS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1alpha,25-(OH)(2)D(3) regulates PKC in GC cells via PI-PLC and PLA(2), but not PC-PLC or PLD, whereas 24R,25-(OH)(2)D(3) regulates PKC in RC cells via PLD2.